Recent studies indicate acetylated-lysine is an under-appreciated post-translational modification with a potentially prominent role in cancer biology. However, despite advances in proteomics, it is still difficult to find lysine acetylation sites in proteins. Although IMAC can be used to isolate phosphorylated peptides in bulk, there is no bulk purification technology for acetylated peptides. The long-term goal of this project is to develop and commercialize a proteomic method for isolating, identifying, and quantifying lysine acetylation sites. This method will contribute to the development of drugs that affect protein acetylation levels in cancer by elucidating their mechanisms of action. It will also identify new acetylation sites that could become targets for cancer diagnosis and treatment. During Phase I we will establish the method and its tools, using an immunoaffinity approach with an antibody specific for acetylated-lysine. We will first screen several acetylated-lysine monoclonal antibodies to select the one that performs best as an immunoprecipitation reagent and that best recognizes acetylated-lysine peptides in a sequence-independent manner. After choosing one antibody for further study, we will isolate and identify acetylated-lysine peptides from several cancer cell lines. Modified peptides will be isolated by a variation of the immunoaffinity technology we developed for phosphorylation profiling and will be identified by commonly practiced liquid chromatography-tandem mass spectrometry methods. To demonstrate that we can evaluate the relevance of the sites we find, we will collect and present information about known and novel human acetylated-lysine sites in an easy-to-use web-based format, as we have done for phosphorylation sites in PhosphoSite(r). This project has the potential to vastly increase the number of known acetylated-lysine sites and to greatly stimulate the newly emerging fields of lysine acetylation and acetylation biology. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]